Determination Of ASFV-Specific T Cell Epitopes And Development Of SARS-CoV-2 MRNA Vaccine

ASFV causes acute,febrile,and hemorrhagic infectious diseases in pigs,leading to huge economic losses in the pig breeding industry.At present,the research of ASFV vaccine is still stagnant.The mechanism of its infection and the specific immune response after infection have not been clearly explained.Studies have shown that ASFV live attenuated vaccine can produce strong protection,which is related to the activation of T cell immune response.However,the safety issue of the use of such vaccines has not been resolved.In addition,the key to developing other types of ASFV vaccines is to screen for protective antigens that can activate T-cell immune responses.As for the screening of ASFV key antigens,many studies were based on ASFV protein immunization of pigs to screen,and it may not obtain for proteins that actually produce protection after ASFV infection.Therefore,the method of screening protective antigens in ASFV-infected surviving pigs is more scientific.In this study,first,we screened the key proteins involved in activating ASFV-specific T-cell immunity,and the results showed that p30,pp62,p72 and CD2 v proteins were all involved in the T-cell immune response of ASFV-infected surviving pigs,and p72 had the highest activation ability.Subsequently,the T cell epitopes of p72 protein were comprehensively scanned,and three high-frequency positive epitopes were identified by ELISPOT using PBMCs of ASFV-infected surviving pigs,and swine leukocyte antigen(SLA)-tetramers were constructed to detect ASFV-specific T cells.Finally,specific T-cell and humoral immunity in ASFV-infected and surviving pigs were compared using SLA-tetramer and ELISA methods.The results showed that ASFV-specific T-cell immune responses gradually increased during infection and peaked in late of infection,and specific T-cell immune responses in surviving pigs decreased;a similar trend was observed in ASFV-specific humoral immune responses,but the humoral immune response of surviving pigs in peaked.In conclusion,our study provides key information for the study of ASFV-specific immune responses and the development of ASFV vaccines.Since the outbreak of COVID-19,it has spread rapidly all over the world,seriously threatening human life and health.Several SARS-CoV-2 vaccines are currently in clinical use,however,variants of SARS-CoV-2 continue to emerge and threaten the protection of existing vaccines.As a novel form of vaccine,m RNA vaccines have been approved for clinical use for the first time in the fight against COVID-19.At present,two m RNA vaccines have been launched,and their Phase III clinical data reports that their effectiveness exceeds 90%,which is the most effective vaccine at present,which proves the advantages of the m RNA vaccine platform.However,the above two m RNAs were developed with S protein as the target protein.However,the above two m RNA vaccines were developed with S protein as the target protein.In view of the reports of ADE phenomenon in SARS-CoV and MERS-CoV vaccines,some studies have shown that receptor binding domain(RBD)as an antigen can produce less of non-neutralizing antibodies.It is safer to develop vaccines with RBD as the target protein.RBD protein has small molecular weight and low immunity,and it is not easy to stimulate immune response,which is one of the problems of its limited use.However,many studies have shown that the development of nanoparticle antigens can greatly improve the antigenicity of proteins.Therefore,in order to increase the antigenicity of RBD,this study selected nanoparticle antigens to display RBD protein to develop SARS-CoV-2 vaccines.In this study,m RNA vaccine encoding the SARS-CoV-2 trimerized receptor binding domain(RBD)protein(T-RBD)and self-assembled nanoparticles formed by trimerizing RBD fused ferritin(TF-RBD)were developed.After immune evaluation in mice,the results showed that TF-RBD produced more robust and durable humoral immunity,as well as a Th1-biased cellular immunity compared with T-RBD.Furthermore,in the SARS-CoV-2 live virus challenge experiment,immunization with two doses of low-dose TF-RBD provided protection to h ACE2-transduced mice.Furthermore,the m RNA template of TF-RBD can be easily and rapidly engineered into SARS-CoV-2 variant vaccine.Finally,the WT and two variants m RNA vaccines were made into multivalent vaccines to immunize mice and the cross-neutralization ability was evaluated in vitro.The results showed that the TF-RBD multivalent vaccine produced broad-spectrum neutralizing antibodies against WT,Alpha(B.1.1.7)and Beta(B.1.351)variant pseudoviruses.Overall,this strategy for designing m RNA vaccines based on self-assembled nanoparticle trimeric RBDs provides a reference for m RNA vaccine research.